一种快速鉴定猪肺炎支原体免疫显性蛋白抗原的ELISA方法的建立

Development of an ELISA for identification of immunodominant protein antigens of Mycoplasma hyopneumoniae

摘要:

旨在建立一种快速鉴定猪肺炎支原体 (Mycoplasma hyopneumoniae，Mhp) 血清体液免疫显性蛋白抗原的方法。通过构建pGEX-6P-1-mhp366重组表达质粒并转入大肠杆菌BL21(DE3)，将GST-Mhp366重组蛋白进行原核表达。将GST-Mhp366重组菌和GST工程菌裂解液加入谷胱甘肽包被板进行抗原包被，分别与17份Mhp阳性血清和13份Mhp阴性血清反应，通过对抗原包被量、封闭液、血清和二抗稀释度的优化，确定间接ELISA的反应条件。最终确定GST-Mhp366重组菌和GST工程菌裂解液原液为抗原的最佳包被量，PBS+10% FBS+2.5%脱脂奶粉为最佳封闭液，分别将血清按照1∶500稀释、酶标二抗按照1∶40 000稀释作为最佳工作浓度，从而建立了一种基于间接ELISA的快速鉴定Mhp血清体液免疫显性蛋白抗原的方法，同时通过已知的血清体液免疫显性蛋白Mhp156和Mhp364对所建立的方法进行了验证。该方法的建立能够用于在基因组水平高通量筛选Mhp血清体液免疫显性蛋白抗原，并为Mhp初乳体液免疫和黏膜免疫显性蛋白抗原鉴定方法的建立奠定基础。

Abstract:

We developed a method to identify serological humoral immunodominant proteinic antigen of Mycoplasma hyopneumoniae (Mhp). After constructing the recombinant plasmid pGEX-6P-1-mhp366 and transforming it into Escherichia coli BL21(DE3), the recombinant GST-Mhp366 protein was expressed successfully. The lysates of the recombinant GST-Mhp366 and genetic engineering GST were added into glutathione coated plates and reacted with 17 positive sera or 13 negative sera. Meanwhile, the optimization of experimental conditions, including coated antigen, blocking buffer, dilutions of sera and second antibody were determined. The optimal concentration of the coated antigen was the original bacteria lysates without dilution, and the optimal blocking buffer contained 10% FBS and 2.5% skim milk in PBS. Besides, the working concentration of serum samples and the HRP-tagged rabbit anti-pig IgG secondary antibody were 1:500 and 1:40 000, respectively. Thus, an indirect ELISA was established for identification of immunodominant protein antigens of Mhp. Meanwhile, this method was confirmed by the identified serological humoral immunodominant proteinic antigen Mhp156 and Mhp364. This method can be used for identification of the candidate vaccine antigens on a genome-wide scale. Furthermore, it can lay the foundation for identifying the candidate vaccine antigens through colostra and the nasal mucosal secretions.